Clinical diagnostic assays now use chemiluminescence as the preferred state-of-the-art detection technology. Assays with chemiluminescent readouts have the most sensitive detection limits and widest dynamic range for analyte quantification. Increasingly, there is a need to design simplified, miniaturized, self-contained, and/or robust diagnostic platforms for use in the field or in developing countries, where standard lab equipment, controlled environments, and technical support are minimal. The current commercially available dioxetane substrates do not fare well where controlled storage conditions are not readily available. Thus, there is a need for a method of generating in situ dioxetane substrates using precursors which have better thermal stability.